Activity-dependent organization of prefrontal hub-networks for associative learning and signal transformation

Associative learning is crucial for adapting to environmental changes. Interactions among neuronal populations involving the dorso-medial prefrontal cortex (dmPFC) are proposed to regulate associative learning, but how these neuronal populations store and process information about the association remains unclear. Here we developed a pipeline for longitudinal two-photon imaging and computational dissection of neural population activities in male mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC network topology. Using regularized regression methods and graphical modeling, we found that fear conditioning drove dmPFC reorganization to generate a neuronal ensemble encoding conditioned responses (CR) characterized by enhanced internal coactivity, functional connectivity, and association with conditioned stimuli (CS). Importantly, neurons strongly responding to unconditioned stimuli during conditioning subsequently became hubs of this novel associative network for the CS-to-CR transformation. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on the activity-dependent formation of the hub network within the dmPFC.

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Source data files are provided with this paper.The data that support the findings of this study are also available from the corresponding author upon reasonable request.The anatomical information in the Allen Brain Atlas (https://atlas.brain-map.org/) was used for the anatomical description, determination of the virus injection area, and evaluation of the recorded brain regions.
Sample sizes (numbers of neurons and animals) were based on work in previous publications (Ref 14,16,41,42), and no statistical tests were used to predetermine sample sizes.
No mice were excluded.Inclusion criteria for recorded neurons were based on the algorithm as described in the previous studies and in the method section of the present study.
Behavioral experimental settings of the fear conditioning, i.e., tones and foot shocks, and those of the two-photon imaging, were used as replicates independently in all mice.To validate the reproducibility and reliability of the obtained results, we tested various parameters and the ways to verify the results as shown in the results (e.g., in Figs.S2, S6-8, and S10), all of which indicated very similar results, specificities, and conclusions.We performed tests based on bootstrap resampling as replicates to evaluate the statistical significance between the two groups systematically.
To verify the neural coactivity measurement, we used not only the original data but also the shuffled data, where the activity of each neuron was preserved but the temporal order was randomly shuffled neuron by neuron.To systematically estimate representative values (e.g.mean or median) of each mouse or each group where the number of recorded neurons in each field view varied, we performed bootstrap resampling as explained in the method section of the present study.
For the behavioral experiments and virus injections, blinding was not relevant because all behavioral experiments were controlled by computer systems, and the virus injections were performed with constant settings and thresholds.The investigators were blinded to group allocation during all data collection and analysis since they were automatically performed with constant settings and thresholds.
imaging and simultaneous behavioral experiments were collected by by Labview 2015/2018 (National Instruments) and FV30S-SW image acquisition and processing software (Olympus) as as described in in the manuscript.